FLUORESCENCE STUDIES OF THE SHAKER POTASSIUM CHANNEL

Shaker是电压依赖性钾(Kv)通道亚家族的模型，它在膜去极化时催化钾离子的跨膜流动。KV通道参与多种生理过程，包括心肌细胞复极和神经元的频率编码。尽管这种完整的膜蛋白已经受到了广泛的生物物理表征，但关于该通道的激活机制的细节还很少。为了阐明跨膜电场影响电压传感器运动的方式，将结合使用光学和电生理技术。利用定点突变和硫醇特定的荧光团，可以通过荧光技术检测特定的结构修饰，并与门控电荷置换相关联。因此，本研究方案将重点研究振荡器通道电压传感器动态特性的两个方面：i.用基于稀土的荧光共振能量转移(LRET)测定S4残基的平衡位置，以及II.利用位点特异性荧光来表征局部构象变化的动力学，这项研究的洞察力将增强离子通道生物物理学的当代观点，并可能导致对正常生理学的更好理解，以及许多通道病的病理生理学。

Shaker, a model for the subfamily of voltage-dependent potassium (Kv) channels, catalyzes the transmembranous flow of potassium ions upon membrane depolarization. Kv channels are involved in a myriad of physiological processes including cardiac cell repolarization and frequency-encoding in neurons. Although this integral membrane protein has been subject to extensive biophysical characterization, details concerning the channel's activation mechanism are scant. In order to elucidate the manner in which the transmembrane electric field affects voltage sensor movement, a combination of optical and electrophysiological techniques will be utilized. The use of site-directed mutagenesis and thiol-specific fluorophores, allows specific structural modifications to be assayed via fluorescence techniques and correlated to gating charge displacement. Therefore, this research proposal will focus on two aspects of Shaker channel voltage-sensor dynamics: i.) determining equilibrium positions of S4 residues with the use of Lanthanide- Based Fluorescence Resonance Energy Transfer (LRET), and ii.) characterizing the kinetics of local conformational changes with the use of site-specific fluorescence The insight gained from this study will augment the contemporary view of ion channel biophysics and may result in a better understanding of normal physiology, in addition to the pathophysiology of numerous channelopathies.